Vlademir Vicente Cantarelli

Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2.

Vibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon‐positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco‐2 and HCT‐8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADP‐ribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS‐secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP‐ribosylates Ras, a member of the low‐molecular‐weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.

Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation.

Enteropathogenic Escherichia coli (EPEC) is able to inject its own receptor, a transmembrane protein called translocated intimin receptor, Tir, into the host epithelial cell. The bacterium then uses an outer membrane protein, intimin, to bind to Tir and remains firmly attached to the host cell surface for the duration of the infection. The bacterium is also able to trigger the rearrangement of several host cell proteins, culminating with the formation of an actin‐rich, pedestal‐like structure beneath the EPEC adherence site. Although several cytoskeletal proteins are rearranged following EPEC infection, the exact role played by these proteins during pedestal formation remains unknown. We report here that talin, an integrin‐binding protein, is recruited by EPEC and associates directly with Tir. By surface plasmon resonance (SPR), the predicted value for the dissociation constant (KD) for Tir–talin binding was 1.86 × 10−7 M. We also demonstrate that microinjection of anti‐talin antibodies into HeLa cells resulted in the complete inability to focus actin filaments beneath the attached bacterium. These findings demonstrate that talin is essential for EPEC‐induced pedestal formation in infected cells.

Interaction of enteropathogenic or enterohemorrhagic Escherichia coli with HeLa cells results in translocation of cortactin to the bacterial adherence site.

ABSTRACT Infection of cultured HeLa epithelial cells with enteropathogenicEscherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) O157:H7 results in accumulation of cortactin under the adherent bacteria. Tyrosine phosphorylation of cortactin is not induced following HeLa cell infection with EHEC or EPEC, contrary to what has been reported to occur with Shigella flexneri.

Cortactin is necessary for F-actin accumulation in pedestal structures induced by enteropathogenic Escherichia coli infection

ABSTRACT Cortactin and the translocated intimin receptor, Tir, interacted with each other in pedestal formation in HeLa cells infected with enteropathogenic Escherichia coli (EPEC). Cortactin is shown to be necessary for organizing actin pedestals in response to EPEC, based on the expression of green fluorescent protein-fused cortactin derivatives in HeLa cells.

Identification of Two Translocon Proteins of Vibrio parahaemolyticus Type III Secretion System 2

ABSTRACT The type III secretion system (T3SS) translocon complex is composed of several associated proteins, which form a translocation channel through the host cell plasma membrane. These proteins are key molecules that are involved in the pathogenicity of many T3SS-positive bacteria, because they are necessary to deliver effector proteins into host cells. A T3SS designated T3SS2 of Vibrio parahaemolyticus is thought to be related to the enterotoxicity of this bacterium in humans, but the effector translocation mechanism of T3SS2 is unclear because there is only one gene (the VPA1362 gene) in the T3SS2 region that is homologous to other translocon protein genes. It is also not known whether the VPA1362 protein is functional in the translocon of T3SS2 or whether it is sufficient to form the translocation channel of T3SS2. In this study, we identified both VPA1362 (designated VopB2) and VPA1361 (designated VopD2) as T3SS2-dependent secretion proteins. Functional analysis of these proteins showed that they are essential for T3SS2-dependent cytotoxicity, for the translocation of one of the T3SS2 effector proteins (VopT), and for the contact-dependent activity of pore formation in infected cells in vitro. Their targeting to the host cell membrane depends on T3SS2, and furthermore, they are necessary for T3SS2-dependent enterotoxicity in vivo. These results indicate that VopB2 and VopD2 act as translocon proteins of V. parahaemolyticus T3SS2 and hence have a critical role in the T3SS2-dependent enterotoxicity of this bacterium.

Two Regulators of Vibrio parahaemolyticus Play Important Roles in Enterotoxicity by Controlling the Expression of Genes in the Vp-PAI Region

Vibrio parahaemolyticus is an important pathogen causing food-borne disease worldwide. An 80-kb pathogenicity island (Vp-PAI), which contains two tdh (thermostable direct hemolysin) genes and a set of genes for the type III secretion system (T3SS2), is closely related to the pathogenicity of this bacterium. However, the regulatory mechanisms of Vp-PAIs gene expression are poorly understood. Here we report that two novel ToxR-like transcriptional regulatory proteins (VtrA and VtrB) regulate the expression of the genes encoded within the Vp-PAI region, including those for TDH and T3SS2-related proteins. Expression of vtrB was under control of the VtrA, as vector-expressed vtrB was able to recover a functional protein secretory capacity for T3SS2, independent of VtrA. Moreover, these regulatory proteins were essential for T3SS2-dependent biological activities, such as in vitro cytotoxicity and in vivo enterotoxicity. Enterotoxic activities of vtrA and/or vtrB deletion strains derived from the wild-type strain were almost absent, showing fluid accumulation similar to non-infected control. Whole genome transcriptional profiling of vtrA or vtrB deletion strains revealed that the expression levels of over 60 genes were downregulated significantly in these deletion mutant strains and that such genes were almost exclusively located in the Vp-PAI region. These results strongly suggest that VtrA and VtrB are master regulators for virulence gene expression in the Vp-PAI and play critical roles in the pathogenicity of this bacterium.

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.

Metallo-β-lactamase-producing Pseudomonas aeruginosa in two hospitals from southern Brazil

This study determined the prevalence of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa in two hospitals located in the Southern part of Brazil and compare the performance of two different phenotypic tests. Thirty-one non-repetitive Pseudomonas aeruginosa isolates from various clinical samples from patients admitted to two hospitals located in Rio Grande do Sul, Brazil (twenty-three from a hospital in Porto Alegre City and eight isolates from a hospital in Vale dos Sinos Region). All strains suggestive of possessing MBLs by phenotypic methods were included in this study. Phenotypic detection of MBLs was carried out simultaneously by using both the MBL Etest and disk approximation test using 2-mercaptopropionic acid close to a ceftazidime disk. Strains positive were further confirmed using molecular techniques for bla(VIM), bla(IMP) and bla(SPM-1). The prevalence of MBLs from samples of inpatients from the hospital located in Porto Alegre was 30.4% and that of inpatients from Vale dos Sinos hospital was only 3.1%. Only MBL type SPM-1 was detected in these samples by molecular analysis and all were detected by the Etest MBL strips. The prevalence of P. aeruginosa that produce MBLs can be markedly different in distinct geographical areas, even among different hospitals in the same area. In our study, the EDTA-based method was the only method able to detect all strains harboring the SPM-1 enzyme.

Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP

Enterohaemorrhagic Escherichia coli (EHEC) is an important food‐borne pathogen that, upon infection, causes destruction of the microvilli brush border of intestinal cells. EHEC is able to recruit several host cell proteins and induce actin accumulation beneath its adherence site, forming a pedestal‐like structure upon which the bacterium is firmly attached. Injection of bacterial effectors into the host cells is required to trigger the recruitment and activation of proteins, such as cortactin, neural Wiskott–Aldrich syndrome protein (N‐WASP) and Arp2/3 complex, directly involved in the actin polymerization process. We found that cortactin, an actin‐binding protein, has a pivotal role during pedestal formation by EHEC. Cortactin was found to bind directly to two important virulence factors of EHEC, Tir and EspFu, which are translocated into the host cells during infection. Binding of cortactin to these effectors is dependent upon tyrosine phosphorylation and a balance between tyrosine phosphorylation and dephosphorylation of cortactin is required to regulate pedestal formation by EHEC.

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal‐like structure beneath the attached bacteria. Using siRNA, we show here that EPEC‐ and EHEC‐induced pedestals are dependent on cortactin, an F‐actin‐binding protein found in the mammalian cell cortex. Knock‐down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F‐actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP–SH3), proline‐rich region (GFP–PRR) or α‐helical region of cortactin markedly reduced the amount of F‐actin at the bacterial attachment sites. Interestingly, neither GFP–SH3 nor GFP–PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the α‐helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted α‐helical region of cortactin in actin assembly induced by EPEC and EHEC.